The objective of this research program is to elucidate, at the molecular level, the mechanism and function of copper binding in the prion protein and the relationship of this interaction to the propagation of prion mediated neurological disease. A misfolded form of the prion protein (PrP) is responsible for a class of fatal neurodegenerative diseases termed the Transmissible Spongiform Encephalopathies (TSEs), which include mad cow disease and Creutzfeldt-Jakob disease in humans. PrP is a membrane bound glycoprotein found in all mammals and avian species. Despite nearly twenty-five years of research on this remarkable protein, its normal physiological function remains unknown. Recent discoveries, however, demonstrate that PrP binds copper with high affinity in its conserved N-terminal octarepeat domain, and that this interaction confers a neuroprotective function of unknown origin. Over the last funding period, the Pi's laboratory identified all relevant Cu(ll) sites, discovered a new site in the region associated with misfolding and provided a detailed molecular characterization of copper occupied full-length PrP. Moreover, it was shown that the Cu(ll) coordination environment changes significantly depending on the precise ratio of copper to protein. The proposed research will build on these discoveries using protein design, biophysical techniques such as electron paramagnetic resonance, and neuronal cell culture. Aim #1 focuses on determining the copper binding affinity and cooperativity in wild type PrP and in mutants with octarepeat expansions associated with disease. Aim #2 will use protein design to select for specific copper binding modes. In turn, these mutants will be tested in cell culture to identify the form of PrP associated with neuroprotective function. Aim #3 will examine how copper influences the assembly kinetics and structure of synthetic prions. These studies will determine whether copper plays a role in disease. Aim #4 will develop of PrP fusion proteins designed to optimize crystallization for X-ray structure determination. The TSEs share pathologies with prevalent, age- related neurodegenerative illnesses such as Alzheimer's and Parkinson's disease. In addition, the potential of prions in food or transmission through medical procedures make the TSEs a continuing health threat. Consequently, this research program will help improve public health and safety, and will provide critical new insights into neurodegenerative diseases associated with aging.